SLO➢ At the end of the class students should be able to

➢ Define erythropoiesis

➢ Describe the steps of erythropoiesis

➢ Describe the regulation of it

➢ Discuss the various blood indices

GENERAL FEATURES• Dimensions

• Normal Count

• Composition

• Functions

• Fragility

• Variations

• Fate & Lifespan

DIMENSIONS

• Shape: Biconcave

• Size: 7.2um in diameter

• Thickness: 2um at the periphery and 1um at the center

• Volume: 87um3

ERYTHROCYTES✓ Surface area is 120µ2.

✓ Normal volume is 80-

94 µ3.

✓ Normal life

span of

an

RBC is 120 days.

ERYTHROCYTESNormal Erythrocyte

Count

Males 5-5.5 million

cells/mm3

Females 4.5-5 million

cells/mm3

Infants 6 – 7 million

cells/mm3

STRUCTURE OF RBC• Lipids: Cholesterol,

phospholipid, andglycolipids

• Proteins: Spectrin,actin, ankyrin.

• The glycolipids:constitute the ABOblood group substances(agglutinogens).

MetabolismOf

RBC

It is met by the glucose metabolism through

the anaerobic Embden- Meyerhof (EMF)

pathway (90%) and the pentose

phosphate shunt (10%).

Advantages of Biconcave Shape of RBCs:

• Greater surface area for exchange of gases.

• Flexibilty of RBC

• Minimal tension when the volume of cell alters.

How is the shape maintained?

➢ Spectrin

- a contractile protein

- maintains shape and flexibility of RBC

- Antigen on cell membrane – helps in blood group classification

COMPOSITION• 62.5% water

• 35% Hemoglobin

• 2.5% :

- Sugar – glucose

- Lipids – Cephalin, Cholesterol & Lecithin

- Protein – Glutathion : insoluble protein which acts as a

reducing agent and prevents damage of hemoglobin

- Enzymes – Carbonic anhydrase and catalase

- Ions – Na+, K+, Ca2+, PO43-

FUNCTIONS

• Respiratory

• Acid Base balance

• Maintain viscosity

• Pigment: various pigments are derived from hemoglobin after

disintegration of RBC.

FRAGILITY AND HEMOLYSIS

• Hemolysis- Breakdown of RBC and liberation of hemoglobin.

• Fragility- Susceptibility of RBC to hemolysis or tendency to

break easily.

• There are 2 types:

1. Osmotic fragility- due to exposure to hypotonic saline.

2. Mechanical fragility- due to mechanical trauma

VARIATIONSPhysiologic causes of increase count:

• Age

• Gender

• High altitude

• Exercise

• Temperature

• Meal

Decrease in count: High barometric pressure, Pregnancy, sleep

Pathological variations:

• Increase: Polycythemia

• Decrease: Anaemia

VARIATIONS IN SHAPE

• Crenation: Shrinkage as in hypertonic solutions.

• Spherocytosis: Globular form as in hypotonic conditions.

• Elliptocytosis: elliptical shape

• Sickle cell: Crescent shape

• Poikilocytosis: Flask, hammer or any other unusual shape.

VARIATIONS IN SIZE

• Physiological conditions: RBC in venous blood slightly larger

than those in arterial blood.

• Pathological conditions:

1. Microcytes – smaller cells

2. Macrocytes – larger cells

3. Anisocytes – cells of different sizes

LIFESPAN AND FATE OF RBC

• Lifespan – 120 days

• Site of destruction:

Reticuloendothelial system

ERYTHROPOIESIS

Hemopoiesis

• Hemo: Referring to blood cells

• Poiesis: “The development or production of”

• The word Hemopoiesis refers to the production & development of all the blood cells:

– Erythrocytes: Erythropoiesis – Leucocytes: Leucopoiesis– Thrombocytes: Thrombopoiesis.

It is the process of development, differentiation and maturation of

RBCs from primitive stem cells

DEFINITION

Theories of erythropoiesis• Monophyletic theory

- Also known as unitary theory.

- There is a common parent cell of all formed elements of blood.

• Polyphyletic theory

- Also known as trialistic theory

- Suggests different group of stem cells gives rise to different blood cells.

Alexander A. Maximow L. Aschoff

Site of Erythropoiesis• During intrauterine life

Mesoblastic stage (3rd week to 3 months)

Hepatic stage (after 3 months)

Myeloid stage (3rd trimester)

Intravascular erythropoiesis

Extravascular erythropoiesis

Nucleated RBCs

Yolk sac Liver & spleen Bone marrow

• In children

- All bones with red bone marrow

- Liver & spleen

• In adults (after 20yrs)

- Ends of long bones like femur, humerus

- Skull

- Vertbrae

- Ribs

- Sternum

- pelvis

PHSC Pluripotent Hemopoietic stem cell

BFU-E (Burst Forming Unit Erythrocyte)

CFU-E (Colony Forming Unit Erythrocyte)

PROERYTHROBLAST

BASOPHILIC ERYTHROBLAST

POLYCHROMATOPHILIC ERYTHROBLAST

ORTHOCHROMATIC ERYTHROBLAST

RETICULOCYTE

ERYTHROCYTEERYTHROCYTE

E

R

Y

T

H

R

O

P

O

I

E

S

I

S

GM CSF erythroIL-1,IL-6,IL-3

GM CSF erythro

PHSC Pluripotent Hemopoietic stem cell

BFU-E (Burst Forming Unit Erythrocyte)

CFU-E (Colony Forming Unit Erythrocyte)

PROERYTHROBLAST

EARLY NORMOBLAST

INTERMEDIATE NORMOBLAST

LATE NORMOBLAST

RETICULOCYTE

ERYTHROCYTEERYTHROCYTE

E

R

Y

T

H

R

O

P

O

I

E

S

I

S

GM CSF erythroIL-1,IL-6,IL-3

GM CSF erythro

Hematopoietic stem cells (HSCs) are bone

marrow cells that are capable of producing all

types of blood cells.

They differentiate into one or another type of

committed stem cells (progenitor cells). These in

turn form the various differentiated types of blood

cells.

There are separate pools of progenitor cells for

megakaryocytes, lymphocytes, erythrocytes,

eosinophils, and basophils; neutrophils and

monocytes arise from a common precursor.

1. STEM CELLS• These cells have extensive proliferative

capacity and also the:

– Ability to give rise to new stem cells (Self Renewal)

– Ability to differentiate into any blood cells lines (Pluripotency)

• Hematopoietic stem cells (HSCs) are bone marrow cells that are capable of producing all types of blood cells.

• They differentiate into one or another type of committed stem cells (progenitor cells).

2. Progenitor cells BFU-E & CFU-E

– BFU-E Give rise each to thousands of nucleated erythroid precursor cells.

– Undergo some changes to become the Colony Forming Units-Erythrocyte (CFU-E)

– Regulator: Burst Promoting Activity (BPA)

• Committed stem cells lose their capacity for self-renewal.

• They become irreversibly committed.

Burst forming unit BFU(E)• Unipotent progenitor cell

• Less sensitive to erythropoietin

• Responds to other stimulus

forms

Colony forming unit CFU (e)• Highly sensitive and dependent on

erythropoietin

ERYTHROPOIESIS

15-20µm- basophilic cytoplasm, nucleus with nucleoli.

14-17µm-mitosis, basophilic cytoplasm, nucleoli disappears.

10-15µm- ’POLYCHROMASIA’Hb appears, nucleus condenses.

7-10µm- PYKNOTIC Nucleus.Extrusion, Hb is maximum.

7.3µm- Reticulum of basophilic material in the cytoplasm.

7.2µm- Mature red cell with Hb.

3. Proerythroblast •15-20 microns

•Nucleus with multiple nucleoli

•Basophilic cytoplasm with perinuclear halo

•No hemoglobin•Mitosis present

4. Basophilic/ early normoblast

• Slight reduction in size 14-17µm

• Large nucleus, nucleoli reduce in number

• Basophilic cytoplasm

• Active mitosis

5. Polychromatophilic/ intermediate normoblast

• 10-15µm size

• ’POLYCHROMASIA’

• nucleus condenses Chromatin lumps

• Hb starts appearing

• Reduced mitoses

6. Orthochromatic normoblast• 7-10µm

•Acidophilic erythroblast which is the last precursor with a nucleus.

• Nucleus is compact & situated near the membrane pyknotic nucleus is extruded

• Cytoplasm is like mature red cell, reflecting a high Hb content.•Mitosis absent

7. Reticulocyte

• Reticular nuclear fragments

• Nucleus extruded

• Slightly larger than RBCs

• The Reticulocyte

– Has no nucleus

– Has no organelles

– Is larger than the mature RBC

– Is not concave

– Has many polyribosomes

– In severe anemia, many of these are released into the blood prematurely→ Reticulocyte response.

– Normally 1% of circulating blood, are reticulocytes.

8. Mature erythrocyte• Reddish, circular,

biconcave cells

• 7-8 µ

• No visible internal structure

• High Hb content

• Bright at centre due to biconcave shape

7.2 µm

Duration of erythropoiesis

HSC to RBC- 21 days

Differentiation phase: from pronormoblast to reticulocyte phase- 5 days

Maturation phase: from reticulocyte to RBC- 2 days

Changes during erythropoiesis

– Decrease in size– Loss of mitotic activity (later part of

intermediate.normo)– Hemoglobinization (intermediate

normoblast)– Change of cell shape (from globular to

biconcave)– Disappearance of nucleus, mitochondria,

RNA, etc– Change of staining (basophilic –

eosinophilic)

Regulation of erythropoiesis❑General factors

- Hypoxia → erythropoietin

- Growth inducers

- Vitamins

❑Maturation factors

- Vitamin B 12

- Folic acid

❑Factors necessary for hemoglobin production

- Vitamin C →Helps in iron absorption (Fe+++ → Fe++)

- Proteins → Amino Acids for globin synthesis

- Iron & copper → Heme synthesis

- calcium, bile salts, cobalt & nickel.

General factors

Hypoxia → erythropoietin

ERYTHROPOIETIN• Glycoprotein MW-34000 (165 AA residues)

Formation

• 85% formed in endothelial cells of the peritubular capillaries of the renal tubules.

• 15% formed in liver, hepatic cells & Kupffer cells.

Breakdown

▪ In liver. Half life is 5hours

ERYTHROPOEITIN Stimuli for production

❖Hypoxia

❖Products of RBC destruction

❖High altitude

❖Anemia

❖Chronic lung or heart diseases

❖Catecholamines

❖Prostaglandins

Androgens

Inhibition

❖ Blood transfusion

Functions of Erythropoietin• Erythropoietin

increases RBC production in 3 ways:

– Promotes pronormoblast production

– Shortens the transition time through the normoblast stage

– Promotes the early release of reticulocytes.

Renal failure

Growth inducers/ Differentiation inducers

• Interleukin 1, 3, 6 (IL-3 is a growth inducer for all cell lines )

• CSF- E (colony stimulating factor – erythro)

Vit B-12

• Source : only animal tissues

• Absorption from ileum

• Functions

• Promotes maturation of RBCs (plays an important role in folic acid synthesis of nucleic acid-DNA)

Sources of vitamin B12

52

Absorption of Vitamin B12 and the role of Intrinsic factor

54Namrata Chhabra

Folic acid

• Green leafy vegetables , yeast, liver

• Function : maturation of RBC

LIFE SPAN OF MEGALOBLAST IS 40 DAYS

57

Pernicious Anemia

Intrinsic factor of Castle- secreted by parital

cells of gastric mucosa

Essential for absorption of Vitamin B12 by

enteric route

Other Factors Regulating erythropoiesisNUTRITIONAL

FACTOR

• Proteins

VITAMINS

• B12 & folic acid – for

synthesis of DNA

• Riboflavin – Normal BM

division

• Pyridoxine – Heme

synthesis

• Vitamin C – absorption of

Fe from gut

MINERALS

• Iron – for Hb

• Cu, Zn, Co– Hb

synthesis

HORMONES

• Testosterone

• Thyroxine, Adrenal

hormones

• Pituitary hormones –

stimulate

Erythropoietin

NEURAL

Stimulation of Hypothalamus

RBC production

Clinical Aspects

Anemias: Reduced RBC count / reduced Hb

concentration

Polycythemia: Increased RBC count

• Polycythemia vera

• Secondary polycythemia- due to hypoxia

FIGURE 10.1: Stem cells. L = Lymphocyte, R = Red blood cell, N = Neutrophil, B = Basophil,

E = Eosinophil, M = Monocyte, P = Platelet.

• Factors necessary for erythropoiesis:

1. General factors:

-Erythropoietin

-Thyroxine

-Vitamins

2. Maturation factors:

-Vitamin B12 (Cyanocobalamin)

-Intrinsic factor of Castle

-Folic acid

HEMOGLOBIN

• Hb is the iron containing coloring pigment of RBC.

• 95% dry weight of RBC; 30 – 34% wet weight.

• Molecular weight of Hb is 68,000

• Normal value:

-At birth: 25g/dl

-From puberty: 14-16 g/dl

-Adult males: 15g/dl

-Adult females: 14.5g/dl

STRUCTURE OF HEMOGLOBIN

• Conjugated protein

• Protein part called Globin and iron containing pigment called

heme.

• Heme part is called porphyrin

and is formed by 4 pyrole rings

• Globin is made up of 4

polypeptide chains – 2 alpha and

2 beta chains.

Types of Hemoglobin:

• Normal: - Adult Hb

- Fetal Hb

• Abnormal derivatives:

-Carboxyhemoglobin

-Methemoglobin

-Sulfhemoglobin

ERYTHROPOIESIS

• The process of origin, development and maturation of

erythrocytes.

➢ Site of erythropoiesis:

• In fetal life: - Mesoblastic stage

- Hepatic stage

- Myeloid stage

• In newborns, children and adults

ERYTHROCYTE SEDIMENTATION RATE

• Red cells have the property of Rouleaux (piling one on the other)

formation.

• Piled red cells are heavier than the individual ones.

• The rate at which the red cells fall is known as ESR.

• Normal values:

Wintrobe’s method: Males: 0 – 9mm/hr

Females: 0 – 20mm/hr

Westergren’s method: Males: 3 – 7mm/hr

Females: 5 – 9 mm/hr

Westergren’s method:

• Westergren’s tube is used which is 300 mm long & opened at

both the ends.

• It requires collecting 2 ml of venous blood into a tube

containing 0 .5 ml of sodium citrate. It should be stored no

longer than 2 hours at room temperature

or 6 hours at 4 °C. The blood is drawn into

the tube to the 200 mm mark. The tube is

placed in a rack in a strictly vertical position

for 1 hour at room temperature,

Wintrobe’s method:

• The Wintrobe method is performed similarly except that the

Wintrobe tube is smaller in diameter than the Westergren tube

and only 100 mm long.

• EDTA anticoagulated blood without extra diluent

is drawn into the tube, and the rate of fall of red

blood cells is measured in millimeters after 1 hour.

PACKED CELL VOLUME

• Hematocrit is the fractional volume of blood that the

erythrocytes occupy

• It is a reliable index of red cell population.

• Normal values : Males – 46%

Females – 42%

RBC INDICES

• MCV (MEAN CORPUSCULAR VOLUME)

– The average volume of single RBC

PCV per 100ml blood

MCV = 10um3

RBC count (million/cu mm)

Normal range : 78 – 94 um3

▪ RBC with normal volume are called Normocytes

▪ RBC with less then normal volume, Microcytes

▪ RBC with more than normal volume, Macrocytes

• MCH (mean corpuscular hemoglobin)

– The average content of Hb in average RBC.

Hb in gm%

MCH = 10pg

RBC count (million/cu mm)

• Normal range: 28 – 32pg

• MCHC (mean corpuscular hemoglobin concentration)

– Express the average concentration of hemoglobin per unit

volume of RBC.

– It defined as the ratio of the weight of hemoglobin to

volume of RBC.

Hb in gm%

MCHC = 100

PCV per 100ml blood

Normal range: 33 – 38 gm/100ml of cells

DISORDERS OF RBC

• Anemia

Morphologic classification

Etiologic classification

• Polycythemia

Polycythemia Vera

Relative polycythemia

Secondary polycythemia

ANEMIA

• Anemia is defined as Hb concentration in blood below the

lower limit of the normal range for the age and sex of the

individual.

• In adults, the lower extreme of normal Hb is taken as 13g/dl

for males and 11.5g/dl for females.

POLYCYTHEMIA

• Abnormal increase in the number of RBCs in the peripheral

blood, usually with increase in Hb level.

• Types:

o Polycythemia Vera

o Relative Polycythemia

o Secondary Polycythemia

POLYCYTHEMIA VERA

• Polycythemia rubra vera / Osler’s disease / Erythremia /

Vaquez’s disease

• Uncontrolled proliferation of erythroid stem cells leading to

excess of erythroid cell mass in the body.

Clinical Features:

• Male predilection – Middle age

• Skin appears flushed, reddened

• Spleen is palpable

Case study

• A 30 yrs old female came to medicine opd

with history of generalized weakness,

pounding of heart on exertion

• Physician examined for pallor which is

present

• Investigation was done:

• Hb-9g/dl

• RBC count:3.5 millions/cubic mm

• What is your provisional diagnosis?